Purpose: The conventional visual acuity (VA) test is not sensitive enough to detect glaucoma macular damage. We aimed to investigate whether VA measurements using low-contrast high-pass optotypes are more sensitive to macular dysfunction in glaucoma and to find the potential structural basis of this difference.Methods: A total of 147 subjects were recruited, including 118 patients with glaucoma (mean age: 46.08 ± 14.64 years) and 29 healthy controls (mean age: 39.83 ± 9.81 years). For each participant, monocular best-corrected VA was measured using a conventional chart and six high-pass charts at 100, 50, 10, 5, 2.5, and 1.25% contrast levels, respectively. The macular retinal thickness and circumpapillary retinal nerve fiber layer (cpRNFL) thickness of all the glaucoma patients were obtained by spectral-domain optical coherence tomography (SD-OCT).Results: Compared with healthy subjects, glaucoma patients with normal vision demonstrated worse VAs in high-pass acuity measurements . Receiver operating characteristic curve (ROC) showed that 1.25% low-contrast high-pass VA was optimal for discriminating between the controls and glaucoma patients . Compared with conventional VA, 1.25% high-pass VA correlated better with nasal-side macular retinal ganglion cell (RGC)-related parameters . There was no difference in the strength of correlations between the VAs measured using different charts and cpRNFL thickness .Conclusions: VA measurement taken with low-contrast (1.25%) high-pass acuity chart is more sensitive in detecting central visual loss in glaucoma than that taken with the conventional chart. Macular RGC damage appears to be associated with low-contrast (1.25%) high-pass visual loss in glaucomatous eyes. Glaucoma is the most frequent cause of irreversible blindness and visual impairment worldwide. It has been projected to affect around 112 million people by 2040 . This peAccumulating evidence shows that macular involvement occurs earlier in glaucomatous eyes than once thought –8. StudiThere are two distinct visual thresholds regarding the conventional black-on-white letters, specifically the detection threshold and the recognition threshold . SignifiVA measured by high-pass filtered optotypes demonstrated higher sensitivity to neural limitations of age-related macular degeneration (AMD) damage than conventional black-on-white letters . HoweverPrevious studies on patients with multiple sclerosis and optic neuritis confirmed that acuity charts with a set of variable contrasts provide qualitatively similar diagnostic information to that provided by the sinusoidal gratings of different contrast and different spatial frequencies in CS tests , 16. PatThis study was performed according to the tenets of the Declaration of Helsinki and was approved by the ethics committee of the Zhongshan Ophthalmic Center (NO.2019KYPJ115). Written informed consent was obtained from all participants prior to the experiment. The subjects were recruited from the Glaucoma Clinic at Zhongshan Ophthalmic Center.The patients with glaucoma in the current study met the following criteria: (1) a diagnosis of primary open-angle glaucoma (POAG) or normal tension glaucoma (NTG) in one or both eyes as determined by at least two glaucoma specialists ; In the 2) at a distance of 4 m.To determine the best-corrected refractive correction and the BCVA of each enrolled eye, a cycloplegic refraction was done on each participant within a week before the experiment. Objective refraction was measured by autorefraction (NIDEK ARK-1) first. Then subjective refinements to achieve the best VA and optimum optical correction were performed using a phoropter (NIDEK RT-5100). The contemplated prescription was then used in a trial frame for monocular VA measurements performed with the ETDRS illuminator cabinets  generated by MATLAB  with the Psychophysics Toolbox for Windows 10, administered on a laptop computer. The e-charts were displayed on a liquid crystal display monitor . Luminance of the display monitor was made linear after gamma correction using a TES-1330A Digital Light Meter . The values of the properties for the display of stimuli in the current study were specified in pixels. The brightness of the screen and the surround luminance was kept consistent. The screen brightness was set at 100%. The room lights were turned off on the side of the screen, ensuring a stable ambient illumination of 8 lux, while the lights on the side of the subjects remained on, providing a luminance of 160 cd/m2) and a darker core (luminance: 3 cd/m2), which formed a constant ratio of 1:2:1 (edge: core: edge), as the optotype design (high-pass design). The mean luminance of the strokes was consistent with the luminance of the gray background (luminance: 112 cd/m2). The contrast was defined as Michelson contrast . The macular images were generated using the “Dense” protocol in high-resolution volume scan mode with an automatic real-time mean value of 15. The imaging covered a 6 × 6 mm area of the macula centered on the fovea. The thickness of each layer was segmented and calculated by the automatic segmentation algorithms of the built-in software (Version 6.3.4). Scans were acquired with 49 B-scans consisting of 1,024 A-scans. All scans were reviewed, and any scan with a quality score <20 dB or segmentation error was excluded from analysis. The ganglion cell complex (GCC) layer thickness was the sum of the thickness of the retinal nerve fiber layer (RNFL), ganglion cell layer (GCL), and inner plexiform layer (IPL); the ganglion cell inner plexiform layer (GCIPL) thickness was the combination of the GCL and IPL see . The aveThe cpRNFL protocol was also conducted, in which 3.4-mm-diameter circle scans were acquired. Sectoral cpRNFL thicknesses, specifically the global average of the circle scan (G), nasal (N), superonasal (NS), superotemporal (TS), temporal (T), inferotemporal (TI), and inferonasal (NI) quadrants, provided by the built-in software were read see .N = 75) and healthy controls (N = 29). Here, normal vision was defined as BCVA equal to or better than 0.00 logMAR on the ETDRS VA chart. The normality of the data was checked using the Shapiro-Wilk test. Bland-Altman plots . Then Fisher's Z transformation was conducted for comparisons of the correlations.Then, the correlations between multiple OCT parameters and VA results were evaluated by Pearson's partial correlation analysis after adjusting for age and spherical equivalent (SE) in all glaucoma participants , the GraphPad Prism statistical analysis package  and MedCalc statistical software .In this part, 20 glaucoma patients with BCVA equal to or better than 0.00 logMAR on ETDRS chart and 20 healthy controls underwent VA tests under the same conditions at two different points in time. The test-retest reliability was analyzed using Bland-Altman plots see .The mean difference between the two tests (the 1st test and the 2nd test) and the 95% limits of agreement (LoA) of each VA chart were calculated separately see .In this section, we tried to determine whether low-contrast high-pass charts are more sensitive for detecting central visual dysfunction in glaucomatous eyes. Thus, we compared the VA data between glaucoma patients with normal vision and healthy controls. A total of 75 glaucomatous eyes with BCVA equal to or better than 0.00 logMAR and 29 healthy eyes were included in the analysis. The characteristics of the participants are summarized in p100% = 0.004, p50% = 0.014, p10% = 0.007, p5% < 0.0001, p2.5% < 0.0001, p1.25% < 0.0001).The differences in VAs measured using conventional chart and each high-pass chart in the glaucoma group and healthy control group are summarized in p100% = 0.0021, p50% = 0.0081, p10% = 0.0012, p5% < 0.0001, p2.5% < 0.0001, p1.25% < 0.0001). As the contrast decreased, the difference between VAs became larger. At the 1.25% contrast level, the slope of the regression line reached −0.88. When we looked at the pattern formed by the data from healthy controls, the difference was relatively constant throughout, and the regression analysis showed no statistical significance .ROC curve analysis was performed to determine the optimal method for discriminating glaucomatous eyes from healthy eyes . The AUCp < 0.001) and superior subfields . Fisher's Z transformation confirmed that high-pass VA at 100, 50, and 5% contrast level demonstrated slightly stronger correlations with some of the nasal-side parameters when compared with that of conventional VA , and 1.25% low-contrast high-pass VA demonstrated stronger structure-function relationships with all of the nasal-side RGC-related parameters .In this section, data from a total of 118 glaucomatous eyes were analyzed. The BCVA was −0.02 ± 0.11 logMAR and the mean deviation (MD) of 30-2 VF was −13.05 ± 8.60 dB. The correlations between OCT parameters and the VAs were examined by Pearson's partial correlation adjusted for age and SE. There were significant correlations between VAs and the overall RGC-related parameters  of macular scans . Among ar = −0.367 to −0.439, pr < 0.05). The conventional VA and high-pass VAs at 100 and 50% contrast settings showed slightly better correlations with the global average cpRNFL thickness . However, Fisher's Z transformation confirmed that there is no difference in the strength of correlations between VAs measured using different charts and cpRNFL thickness .The correlations between cpRNFL parameters and the VAs are summarized in Glaucoma has gradually become known as a condition that has macular involvement in the early stage even with well-preserved VA , 7, 21, Shah et al. confirmed that in foveal viewing, while conventional letters are good stimuli for detecting defocus, high-pass filtered letters were less vulnerable to optical defocus and more sensitive to neural limitations in conditions such as AMD , 25. A sIn the present study, we sought to measure the effect of both a high-pass design and a low-contrast setting on the pattern resolution of glaucomatous eyes under foveal viewing. We wanted to first confirm whether low-contrast high-pass optotypes could better serve as stimuli for glaucoma detection and then to assess the structure-function relationships between retinal thicknesses measured by SD-OCT and VAs to find the potential structural basis that undermined central pattern vision in glaucomatous eyes.In agreement with previous studies , 25, ther = −0.363 to −0.410), and the global average cpRNFL thickness showed the highest correlation with coefficient value of −0.447 . The patients/participants provided their written informed consent to participate in this study.YW, ZC, and MY conceived and designed the study. ZC, YK, and HC conducted the coding and programming. YW, ZC, CZ, YY, JX, and MY analyzed and interpreted the patient data. YW analyzed the data and was a major contributor in writing the manuscript. All authors provided critical appraisal and final approval of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Indoleamine 2,3-dioxygenase 1 (IDO-1) is an immunosuppressive enzyme expressed in the placenta, neoplastic cells, and macrophages to reject T cells by converting tryptophan into kynurenine. However, the role of IDO-1 in brain immunity, especially in the meninges, is unclear. We aim to elucidate the distribution pattern of IDO-1+ macrophages/microglia in the human brain tissues, human glioblastoma, APP/PS1 mouse brains, and quinolinic acid model brains and explore the physiological and immunological roles of IDO-1+ macrophages/microglia. Here, we find that both human and mouse macrophages/microglia of the perivascular and subarachnoid space and in glioblastoma (GBM) expressed IDO-1 but not macrophages/microglia of parenchyma. Using IDO-1 inhibitors including 1-MT and INCB24360, we observed that inhibiting IDO-1 reduced the cellular size and filopodia growth, fluid uptake, and the macropinocytic and phagocytic abilities of human blood monocytes and RAW264.7/BV-2 cells. Inhibiting IDO-1 with 1-MT or INCB24360 increased IL-1β secretion and suppressed NLRP3 expression in RAW264.7/BV-2 cells. Our data collectively show that IDO-1 expression in perivascular and meninges macrophages/microglia increases cellular phagocytic capacity and might suppress overactivation of inflammatory reaction. Indoleamine 2,3-dioxygenase 1 (IDO-1), an immunosuppressive metabolic enzyme, prevents maternal T cell-driven immune rejection during pregnancy by metabolizing tryptophan into kynurenine . Meninge-/- mice also secrete type I IFN in LP-BM5 infection and restrict viral replication Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: PartlyReviewer #3: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: NoReviewer #3: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The manuscript by Rong Ji and colleagues titled “Characterization of IDO-1 expressing macrophages/microglia in the meninges and perivascular space of the human and murine brain” described the role of Indoleamine 2,3-dioxygenase 1 (IDO-1) enzyme in supporting the activities of the macrophages/microglia in meninges using human and murine brain samples. The authors were also explained the physiological and immunological role of IDO-1+ macrophages/microglia after treating with 1-MT and INCB2436, inhibitory molecules in murine and human models using immunofluorescence staining, cell migration and proliferation assays. The real time PCR and western blot analysis were used to support the data. If the author would have added other cell proliferation assays and flow cytometry data (if available), that would have been more effective to prove their hypothesis. The manuscript may be appropriate for the publication after a major revision and answering the following comments.Major comments:A)Abstract-1. In the line 3- says” Indoleamine 2,3-dioxygenase 1 (IDO-1) expressed in macrophages rejects T-cells”. It is not clearhow the IDO-1 rejects T-cells? It would be good if the author explains with some more examples.2. Line 6: disease models- please specify which diseases models are you mentioning here?3. Line 10: 1-MT and INCB2436- It would be convenient for the readers if the author states clearly about these inhibitorymolecules.B) Introduction-4. it is specified that “limitans are thought to contribute to T-cell rejection”. It is not clear is it immune mediated T-cellrejection or inhibition of migration of T-cells. How do you explain the T-cells rejection? Hypothesis may be reformedclearly.5. The sentence “IDO-1 does not drive the formation of M1 macrophages” contradicts your own statements which says IDOis expressed in M1s. It would be more clear if the author could rewrite the introduction with suitable references.C) Materials and Methods:6. Samples details from the human GBM patients are not available. In order to analyze in all possible aspects, moreinformation on the age or gender of the human patients may be included.7. The passage numbers and the source/ATCC equivalent details of the cell lines RAW264.7 and BV-2 cells may beincluded.8. Real time PCR: please specify what is the limit of detection, or limit of quantification and the PCR efficacy of the Realtime RT-PCR used in the study.9. The procedure for Interleukin ELISA is missing. Brief procedures for the ELISA may be included.10. Please specify the statistical tests used in the study in the method section.D) Results and discussion:11. In the Fig legends the human and mouse brain samples were not clearly specified. Please specify the tissue of origin.12. In Fig legend 1. Explanation for Fig 1E is missing. Is it the magnified region of Fig 1D? is it neoplastic lesions or normalbrain tissues?13. Have you tested the migration and proliferation of both M1 and M2 macrophages along with 1-MT and INCB2436treatment? If so discuss the results in comparison with each types.14. The possible explanation on the differential effects of 1-MT, INCB24360, and IFN-g treatments on proliferation andmigration ability of the M1 or M2 macrophages need an elaborated in discussion.15. The discussions need to be re written adding more references on the functional effect of the IDO-1. At the same timethe shortcomings of the study and the future prospects of your study need to be mentioned.Minor comments:1. In abstract - Line 15: IDO-1 expression in perivascular macrophages instead of IDO-1 in macrophages.2. There are some lines in the text where IDO-1+ has been mentioned, it would be convenient to mention IDO-1expressing cells.3. In Fig 2 the units of the graphs need to be mentioned as fold change or log change.4. Abstract Line 7: “Here” - Please specify is this at the steady state? Human or mouse?5. The primer details of the house keeping gene, alpha-tubulin is missing.Reviewer #2: The current study presents intriguing finding in the brain immunology. The authors propose that IDO-1+ macrophages/microglia in meninges have higher scavenging ability which might be involved in preventing meninges/perivascular space T cells from entering the parenchyma maintaining immunosuppressive environment in the brain. While the manuscript represents an interesting finding and has a decent amount of immune fluorescence evidences and data, there are major considerations that prevents the manuscript’s publication in its current form.Major Considerations:1- The major issue in this manuscript is the lack of enough quantitative evidences to support the data. The main analysis in the study was based on manual morphometric analysis of the immune fluorescent photos. While this is a valid analysis, it is subjective and require further verification with other studies “when cells are manually selected, a heavy operator‐dependent bias is introduced, impairing procedure reproducibility ”.• Fig.1A, the control photo shows extensive microglia/macrophage  raising a question about the health of the brain tested and quality of the preparation (high background noise).• Fig 1C, the authors need to show higher magnification of the cells without the red filter similar to the others.• Fig. 1E,  IDO-1+ cells are negative for IBA-1. How the authors explain these findings? Although, it is reported that macrophages form about 30-50% of inflammatory infiltrate in the tumor mass and they are known to be IDO+?(Sevenich 2018)• Fig.3A, the authors measured the control cells’ filopodia. While they choose the upper cells to measure, another type of cells with extensive processes could be visualized in the same filed. What is the rationale that authors used in selecting the cells for analysis?• The authors need to add the negative control for the staining to exclude non specific staining, for example the neurons in the same field should be negative for both staining.• Fig. 3A, the authors concluded on the polarization ratio between M1&M2 using morphology as a differentiating point. While there is reported difference in the morphology between M1&M2, the inverted phase morphology is not enough as the accuracy of the morphological differentiation between the two cells is based on differences in size, perimeter, shape, intensity, and texture of the actin and nuclear stain , which wouldn’t be achieved by the inverted phase light microscope. Accordingly, other methods like phenotypic marker expression , cytokines analysis should be used for differentiation the two types. Also, quantitative measurement of the M1/M2 ratio using pan-macrophage markers staining and analysis by flowcytometry would have given more precise data .• Fig. 7, the authors are reporting on the dextran uptake and latex beads phagocytosis. While the photos are representative, we suggest integration of the results with flowcytometry uptake studies.• Fig.2&5, while the cytokines’ mRNA expression is a valid representative way, including supernatant’s protein analysis using ELISA, similar to supplementary figure 7, could add to the results.2- The statistics needs to be revised thoroughly through the study, for example:• Fig.2B , there is a visible difference in the mean between control and 1-MT, yet it does not show statistical significance. This could be explained by small sample size, or unequal variability between the groups or non-normal distribution of the data. Accordingly, optimum sample size >6, blotting of the individual values and confirming the validity of the statistical test can solve this.• Fig.3A, How the authors explain the variability between the sample size between groups in the same comparison? And how One way ANOVA was conducted with missing values? , while the legend states that n=7. This again was observed in Fig.3B, Fig.5A and Fig.6A.• How many times the experiments were repeated?• At figure 6A, the authors stated that n>5 without stating what is the sample size exactly and if there is discrepancy between groups. While this is a valid way to represent sample size, however if you are going to statistically compare between them using One Way ANOVA, the exact sample size in each group should be reported. This again was observed in Fig. 4,5 and 7.To solve the previous issues, meticulous revision needs to be done to the figures and the legends. I also recommend using dot plot figure whenever possible instead of bar graph to show the individual values and adding the mean and the SEM values in the text specially with the polarization ratios.3- A previous study in 2012 has found that “IDO Expression in Brain Tumors Increases the Recruitment of Regulatory T Cells and Negatively Impacts Survival”. Based on this study findings, how the authors can explain this? Minor Considerations:1- Please review the references (1-4) as they do not match the text in the first paragraph. While the text mentions on IDOs, these references describe the microglia role in Parkinson’s disease and the polarization of macrophage.2- In the legend of figure 1, the authors need to describe which is murine brain and which is human brain as the figures should be able to stand by itself.3- Figure 1E is not explained either in the results section nor the figure legend.4- Fig. 2, the statistical significance was not demonstrated in some graphs.5- The manuscript needs to be revised for typo error like• Page 17 line 14, However However (delete one of them)• Page14 line 20, Adipogenic? (Should be Adipogen?)References:Antonios, J. K., Z. Yao, C. Li, A. J. Rao and S. B. Goodman (2013). "Macrophage polarization in response to wear particles in vitro." Cellular & Molecular Immunology 10(6): 471-482.Rostam, H. M., P. M. Reynolds, M. R. Alexander, N. Gadegaard and A. M. Ghaemmaghami (2017). "Image based Machine Learning for identification of macrophage subsets." Scientific Reports 7(1): 3521.Ruffinatti, F. A., T. Genova, F. Mussano and L. Munaron (2020). "MORPHEUS: An automated tool for unbiased and reproducible cell morphometry." Journal of Cellular Physiology 235(12): 10110-10115.Sevenich, L. (2018). "Brain-Resident Microglia and Blood-Borne Macrophages Orchestrate Central Nervous System Inflammation in Neurodegenerative Disorders and Brain Cancer." Frontiers in Immunology 9(697).Wainwright, D. A., I. V. Balyasnikova, A. L. Chang, A. U. Ahmed, K.-S. Moon, B. Auffinger, A. L. Tobias, Y. Han and M. S. Lesniak (2012). "IDO Expression in Brain Tumors Increases the Recruitment of Regulatory T Cells and Negatively Impacts Survival." Clinical Cancer Research 18(22): 6110.Reviewer #3: In this manuscript Ji et al investigated differential expression of IDO1 in microglia cells in the brain paranchyma, meninges, brain tumors and brain injury models. In addition, they also investigated potential function of IDO1 in microglia/macrophages. Below are some of the concerns that need to be addressed.1. Throughout the manuscript, please improve the writing. Logically explain why an experiment was done and what is the conclusion2. Rephrase the concluding paragraph in the introduction- it is confusing and hard to understand.3. Please explain why INF-Gamma was used- it is kind of choppy and it appears out of nowhere.4. Please include genetic inhibition of IDO1 and carry out some of the phenotypic data to provide additional evidence for IDO1 functions in microglia.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  7 Jun 2021Response to editor-Dr. Nagaraj Kerur and reviewersDear Dr. Saiyin,Thank you for your patience while the manuscript was being evaluated by the reviewers. We now have evaluation by three reviews. As you see below the reviewer have unanimously recognized the significance of the work presented, however several serious technical and conceptual concerns have been raised. We ask you to address the reviewers’ comments, particularly those related to quality of the immunofluorescence data. All immunofluorescence images should be supported by appropriate positive and negative controls by including isotype antibody staining. Additional the manuscript needs significant improvement in narrative as pointed out by the reviewers. It would be helpful to present your work and describe your data in the context of existing literature about IDO-1 in brain microglia. Additionally, RAW264.7 and BV-2 cells are not good surrogate for brain microglia cells, studies in these cells should reproduced in primary mouse/human microglia.https://www.mybiosource.com/monoclonal-human-antibody/ido-1/303376). iNOS, CD206 that we used in this paper have plenty of publications to support the specificity of these antibodies both in immunostaining and western blotting analysis. Our immunostaining system has a clean background in mouse brain .Response to the editor: Thanks for your kind comments. All staining has positive and negative control. As we are from a medical university, we are good at immunostaining and quality controls of staining. We have selected qualified and well-tested antibodies during staining. In addition, we have used multiple human tissues to testify the specificity of our antibodies. IDO-1 antibody is tested in pancreatic cancer tissues and liver cancer, and multiple cell lines. The IDO-1 staining patterns in pancreatic cancer are consistent with other works in pancreatic cancer and immunohistochemistry database  . These observations further support our findings in this work.Response to the reviewerComments to the AuthorReviewer #1: The manuscript by Rong Ji and colleagues titled "Characterization of IDO-1 expressing macrophages/microglia in the meninges and perivascular space of the human and murine brain" described the role of Indoleamine 2,3-dioxygenase 1 (IDO-1) enzyme in supporting the activities of the macrophages/microglia in meninges using human and murine brain samples. The authors have also explained the physiological and immunological role of IDO-1+ macrophages/microglia after treating with 1-MT and INCB2436, inhibitory molecules in murine and human models using immunofluorescence staining, cell migration, and proliferation assays. The real time PCR and western blot analysis were used to support the data. If the author would have added other cell proliferation assays and flow cytometry data (if available), that would have been more effective to prove their hypothesis. The manuscript may be appropriate for the publication after a major revision and answering the following comments.Thanks for raising these questions. We found that IFN-γ treatment dramatically increased the cellular size. 15-25% of IFN-γ treated cells reached 50-80µm in diameter, some of them reached 100µm in diameters. We have consulted our core facility technician, and they informed us that the size of NOZZLE of Flowcytometry in the core facility is 100µm. The general rule of thumb is that your nozzle size should be about 4-5 times larger than the size of the cells being interrogated. Theoretically, our flow cytometry only analyzes the cell that is smaller than 33µm in diameters. If we analyze a cell with 50-80µm in diameters, we need a specialized NOZZLE. Unfortunately, our core facility is not equipped with a NOZZLE larger than 100µm. Thus we have used staining to evaluate the cells.Major comments:A) Abstract-1. In the line 3- says" Indoleamine 2,3-dioxygenase 1 (IDO-1) expressed in macrophages rejects T-cells". It is not clear how the IDO-1 rejects T-cells? It would be good if the author explains with some more examples.Response 1-1: Thanks for pointing this out. To limit the space, we have shortened the sentence. IDO-1 metabolizes tryptophan into kynurenine, resulting in depleting tryptophan and producing immune-suppressive kynurenine that recruits Tregs and MDSC. Tregs and MDSC suppressed cytotoxic T cell activity. We have shortened the sentence in the abstract. 2. Line 6: disease models- please specify which diseases models are you mentioning here?Response 1-2: Thanks for pointing this out. We specified disease models in the revised version.3. Line 10: 1-MT and INCB2436- It would be convenient for the readers if the author clearly states these inhibitory molecules.Response 1-3: Thanks for pointing this out. We have clearly stated that in the revised version.B) Introduction-4. it is specified that “limitans are thought to contribute to T-cell rejection”. It is not clear is it immune mediated T-cell rejection or inhibition of migration of T-cells. How do you explain the T-cells rejection? Hypothesis may be reformed clearly.Response 1-4: Thanks for pointing this out. The question that you have raised is in the scale of our future work. Based on current understanding, glia limitation is a physical barrier that bars the T cells into the parenchyma. However, this explanation did not include the possibility of the existence of immune cell roles. In this work, we discuss the physiological and immunological roles of IDO-1+ macrophage/microglia in vitro and to what extent IDO-1 expression affects the behaviors of macrophages. In the future, we wish to explore the roles of IDO-1+ macrophage/microglia. 5. The sentence “IDO-1 does not drive the formation of M1 macrophages” contradicts your own statements which says IDO is expressed in M1s. It would be more clear if the author could rewrite the introduction with suitable references.Response 1-5: Thanks for pointing this out. This is an inadvertent mistake. We have deleted itC) Materials and Methods:6. Samples details from the human GBM patients are not available. In order to analyze in all possible aspects, more information on the age or gender of the human patients may be included.Response 1-6: We have done IF in 4 patients. We have added the patients’ information in supplementary materials (Please refer to supplementary table-1). As the size is smaller than 10, we thought that the analysis of relationships between clinicopathological characteristics and IDO-1 is difficult to provide insightful information. 7. The passage numbers and the source/ATCC equivalent details of the cell lines RAW264.7 and BV-2 cells may be included.Response 1-7: Thanks for pointing this out. After 5-6 passages, the RAW264.7 and BV-2 cells were used for experiments. The RAW264.7 cells were purchased from Applied Biological Materials Inc. . The BV-2 cells were purchased from China Center for Type Culture Collection. 8. Real time PCR: please specify what is the limit of detection, or limit of quantification and the PCR efficacy of the Real time RT-PCR used in the study.Response 1-8: Thanks for raising this question. We thought that the limit of detection is not the scope of our study. We all know that transcription levels of one gene correlated with protein levels sometimes in cells or tissues; other times, the transcription levels of one gene do not reflect the protein levels in cells or tissue. Thus we used IF and WB to strengthen our arguments. 9. The procedure for Interleukin ELISA is missing. Brief procedures for the ELISA may be included. Response 1-9: Thanks. We have added this part to the materials and method section in the revised version . 10. Please specify the statistical tests used in the study in the method section.Response 1-10: Thanks for pointing this out. We have specified the statistical tests in the revised version.D) Results and discussion:11. In the Fig legends the human and mouse brain samples were not clearly specified. Please specify the tissue of origin. Response 1-11: Thanks. We have specified the origin of tissue in the revised version.12. In Fig legend 1. Explanation for Fig 1E is missing. Is it the magnified region of Fig 1D? is it neoplastic lesions or normal brain tissues? Response 1-12. This is an inadvertent labeling mistake. Sorry for bring confusion. We corrected it.13. Have you tested the migration and proliferation of both M1 and M2 macrophages along with 1-MT and INCB2436 treatment? If so discuss the results in comparison with each types.Response 1-13: Thanks for pointing this out. We did not test the migration and proliferation of both M1 and M2 macrophages along with 1-MT and INCB2436 treatment. The migration and proliferation of both M1 and M2 macrophages in 1-MT and INCB2436 treatment not the prominent scope of this work.14. The possible explanation on the differential effects of 1-MT, INCB24360, and IFN-g treatments on proliferation and migration ability of the M1 or M2 macrophages need an elaborated in discussion. Response 1- 14: Thanks for raising these questions. We have discussed this in the revised version. “Consistent with a lower migrating ability of M1 macrophages and morphometric analysis, IFN-γ treatment significantly reduced the migration of RAW264.7 and BV-2 but not 1-MT, INCB24360. The multiple filopodia of macrophage in IFN-γ treatment might contribute to the phagocytic or endocytic ability but not migrating”. 15. The discussions need to be re written adding more references on the functional effect of the IDO-1. At the same time the shortcomings of the study and the future prospects of your study need to be mentioned.Response 1-15: Thanks for your suggestions. Similar to the reviewer #2 views, this topic is really compelling for us. We all might have many chances to expand the topic based on our observation. In the future, to what extent IDO-1 expressing microglia/macrophages contribute to immune barriers of the brain parenchyma and if the IDO-1 expressing endothelial cells also contribute to the rejection of T cells by parenchyma? is worth exploring. Another interesting point is that if IDO-1 deregulation in macrophage/microglia correlated with brain autoimmunity diseases caused by the abnormal entrance of T cells to parenchyma, such as Multiple Sclerosis .Minor comments:1. In abstract - Line 15: IDO-1 expression in perivascular macrophages instead of IDO-1 in macrophages. [please replace it]Response 1-16: Thanks. We did it.2. There are some lines in the text where IDO-1+ has been mentioned, it would be convenient to mention IDO-1expressing cells.Response 1-17: Thanks for pointing this out. We replaced it3. In Fig 2 the units of the graphs need to be mentioned as fold change or log change.Response 1-18: Thanks. The unit fold change. We added it in the revised version.4. Abstract Line 7: “Here” - Please specify is this at the steady state? Human or mouse?Response 1-19: Thanks. We have specified in the revised version.5. The primer details of the house keeping gene, alpha-tubulin is missing.Response 1-20: Thanks. Our house keeping gene in RT-PCR is GAPDH. We included the primer of “GAPDH” in previous version. Alpha-tubulin was only used for western blotting.Reviewer #2: The current study presents intriguing finding in the brain immunology. The authors propose that IDO-1+ macrophages/microglia in meninges have higher scavenging ability which might be involved in preventing meninges/perivascular space T cells from entering the parenchyma maintaining immunosuppressive environment in the brain. While the manuscript represents an interesting finding and has a decent amount of immune fluorescence evidences and data, there are major considerations that prevents the manuscript’s publication in its current form.Thanks for your insightful comments. Meninges immunity is intriguing for us as you, especially IDO-1+ microglia/macrophage. Dr. Lili provides us some samples of the freshly surged unaffected brain from glioma patients, making it possible to see the distribution pattern of IDO-1+ macrophages in the human brain. We are willing to move forward as quickly as possible. Unfortunately, COVID-19 changed everything, especially our lab work and students' accessibility to lab and animal models. Our work stopped for near eight months. Here we provided and shared this evidence to the scientific community to push this work forward. We know that in vivo works will provide vital pieces of evidence to IDO+ macrophages in brain immunity. We hope our data are enough to support the conclusion in this paper. Major Considerations:1- The major issue in this manuscript is the lack of enough quantitative evidences to support the data. The main analysis in the study was based on manual morphometric analysis of the immune fluorescent photos. While this is a valid analysis, it is subjective and require further verification with other studies "when cells are manually selected, a heavy operator‐dependent bias is introduced, impairing procedure reproducibility ”.Response #-2-1: Thanks for raising this question. We agree that on manual morphometric analysis exist some bias as other analysis. The reason we used morphometric analysis is that we found that IFN-γ treatment dramatically increased cellular size. 15-25% of IFN-γ treated cells reached 50-80µm in diameter, even though they reached 100µm in diameters. We have consulted our core facility technician. They informed us that the size of NOZZLE in core facility Flowcytometry is 100µm. The general rule of thumb is that your nozzle size should be about 4-5 times larger than the size of the cells being interrogated. Theoretically, our flow cytometry only analyzes the cell that is smaller than 33µm in diameters. If we analyze a cell with 50-80µm in diameters, we need a specialized NOZZLE. Unfortunately, our core facility is not equipped with a NOZZLE larger than 100µm. We have analyzed the dextran uptake in RAW264.7 by Flow after treatment. The trendy of data is nearly consistent to our results . Thus, we believe that our manual morphometric analysis is reliable. • Fig.1A, the control photo shows extensive microglia/macrophage  raising a question about the brain's health tested and quality of the preparation (high background noise).Response #-2-2: Thanks for raising this question. We have stained a brain section 50µm thick. Thick sectioned staining and Z-stack construction often visualize more processes and cells. As autofluorescent lipofuscins widely exist in the adult human brain, you need to be careful and well-trained when you read thick sections. We have sectioned some thin sections and further done H&E staining of the thin slice to confirm tissue status before our staining. If you wish to see the status of tissues, we are willing to provide them. .• Fig 1C, the authors need to show higher magnification of the cells without the red filter similar to the others.Response #-2-3: Thanks for raising this question. We showed a higher magnification insert without a red filter in the revised version. • Fig. 1E,  IDO-1+ cells are negative for IBA-1. How the authors explain these findings? Although, it is reported that macrophages form about 30-50% of inflammatory infiltrate in the tumor mass and they are known to be IDO+? (Sevenich 2018)Response #-2-4: Thanks for pointing this out. We inadvertently labeled E as F in the figure legend. We are sorry for bringing confusion. Here we corrected the inadvertent mistake in the revised version. You are right, and we observed plenty of IDO+ macrophages in pancreatic cancer, GBM, and HCC (Please refer to Editor Reviewers only figure-2). In this study, we only focused on the functions of IDO+ macrophage/microglia in physiological conditions. In the future, we will discuss the role of IDO+ macrophage in pancreatic cancer and GBM. In this part, we used the IDO+ neoplastic cells of GBM as a positive control. The academic editor also raises question about controls in immunostaining. Please refer to the reviewer and editor Fig.2-3.Although, it is reported that macrophages form about 30-50% of inflammatory infiltrate in the tumor mass and they are known to be IDO+? (Sevenich 2018)Thanks for raising this question. This is a big question. It is a little far from the scope of this paper. Like other tumors, brain tumors have plenty of immunes infiltrates. Based on our knowledge, the complicated network of immune cells in tumors needs more time to address by delicate and rigorous work. If we have a chance, we will do it. We wish not to include the topic of tumor in this paper [please refer to the reviewer and editor Fig.2 and Fig.3]. Thanks for understanding. • Fig.3A, the authors measured the control cells' filopodia. While they choose the upper cells to measure, another type of cells with extensive processes could be visualized in the same field. What is the rationale that authors used in selecting the cells for analysis?Response #-2-5: Thanks for pointing this out. It seems that the reviewer is confused by our data. The magnified region only showed typical cellular filopodia in the macrophages in each group. The lower panel is from the upper panel, and the channel is shown as a gray color. As gray is easy to read, we changed the display pattern. The representative image does not mean that we only counted the cells; the representative image is only the image that the author thought it is typical of this group. We have counted a group of the image in our previous version. In the revised version, we have increased the counts. • The authors need to add the negative control for the staining to exclude nonspecific staining, for example the neurons in the same field should be negative for both staining.Response #-2-6: This question was also raised by the academic editor. In our human brain's thick section staining system, it is difficult to do a reasonable control as in thin slide . The adult human brain neurons accumulate lipofuscins, autofluorescent particles in the neuron excited by 405, 488, and 555 lasers. However, we did not notice that large or decent lipofuscin in the microglia and astrocytes in the human brain. We have used well-tested antibodies in our research. I am a histologist who has more than 20-years’ experience in histology. We have tested our antibodies in wide range of tissues. .• Fig. 3A, the authors concluded on the polarization ratio between M1&M2 using morphology as a differentiating point. While there is reported difference in the morphology between M1&M2, the inverted phase morphology is not enough as the accuracy of the morphological differentiation between the two cells is based on differences in size, perimeter, shape, intensity, and texture of the actin and nuclear stain , which wouldn’t be achieved by the inverted phase light microscope. Accordingly, other methods like phenotypic marker expression , cytokines analysis should be used for differentiation the two types. Also, quantitative measurement of the M1/M2 ratio using pan-macrophage markers staining and analysis by flowcytometry would have given more precise data .Response #-2-7: The living cells are versatile. The morphology of the same cells differs from lab to lab based on their culturing system and cellular density also dramatically affects cellular morphology. Moreover, classifying macrophage by its morphology is debated for many years . However, someone did an excellent job based on morphology . I am a histologist who has done morphological analyses for more than 20 years. We all know that the morphology of cells is still the golden standard in histology and pathology despite versatile. This study has done a good control on cellular density in the dishes and culturing system [testing infection]. Our cellular density is lower than 40% confluent in the culturing system. We used inverted microscopy to analyze the morphology, but we have applied confocal microscopy to 3D scan the cellular morphological changes after Phalloidin staining . In addition, cellular migration assay data validate our morphological analysis . As the methodology preference differs from lab to lab and group to group, we wish that the reviewer accepts our morphological analysis preference.• Fig. 7, the authors are reporting on the dextran uptake and latex beads phagocytosis. While the photos are representative, we suggest integration. of the results with flowcytometry uptake studies.Response #-2-8: Again. The reason we used morphometric analysis is that we found that IFN-γ treatment dramatically increased cellular size. 15-25% of IFN-γ treated cells reached 50-80µm in diameter, even though they reached 100µm in diameters. We have consulted our core facility technician. They informed us that the size of NOZZLE in Flow cytometry at core facility is 100µm. The general rule of thumb is that your nozzle size should be about 4-5 times larger than the size of the cells being interrogated. Theoretically, our flow cytometry only analyzes the cell that is smaller than 33µm in diameters. If we analyze a cell with 50-80µm in diameters, we need a specialized NOZZLE. Unfortunately, our core facility is not equipped with a NOZZLE larger than 100µm. In the revised version, we used Flow cytometry to analyze dextran uptake. The trendy of data is nearly consistent to our results . • Fig.2&5, while the cytokines’ mRNA expression is a valid representative way, including supernatant’s protein analysis using ELISA, similar to supplementary figure 7, could add to the results.Response #-2-9: Thanks for pointing this out. Our manuscript is mainly focused on the physiological and immunological roles of IDO-1+ upregulation in microglia/macrophages. M1 and M2 are effective way to classify the physiological and immunological roles. However, this classification is controversial sometimes. Including supernatant’s protein analysis using ELISA in Fig.2&5 will strengthen our data. However, we thought that our data is enough to support our conclusion in this paper. This is a specific season, and we are willing to prepare more data for our paper. Unfortunately, you might not know the university is in a partially open condition. Thanks for your understanding.2- The statistics needs to be revised thoroughly through the study, for example: • Fig.2B , there is a visible difference in the mean between control and 1-MT, yet it does not show statistical significance. This could be explained by small sample size, or unequal variability between the groups or non-normal distribution of the data. Accordingly, optimum sample size >6, blotting of the individual values and confirming the validity of the statistical test can solve this.Response to reviewer #2-10. Thanks for pointing this out and gave us useful suggestions. We have repeated RT-PCR of CD206 and Arginase 1 (Arg1) gene and added this part to the paper. Based on your suggestions, we have modified the statistical plots of CD206 and Arg-1. We have done RT-PCR to see the transcriptional changes. As most protein levels data are consistent with the transcription changes, we thought three repeats are enough. We thought the strength of our recent data is enough to support the conclusion in this manuscript. Thanks for understanding. • Fig.3A, How the authors explain the variability between the sample size between groups in the same comparison? And how One way ANOVA was conducted with missing values? , while the legend states that n=7. This again was observed in Fig.3B, Fig.5A and Fig.6A.Response to reviewer #2-11：1) Thanks for raising these questions. As it is almost impossible that the cells are evenly distributed in the culturing dishes, the number of cells varied from image to image. In addition, some cells huddled together in some regions, which makes them difficult to identify and count. Thus, some figures have more countable cells than others, which causes the differences of n. We counted 500-800 cells in each group, the statistical power of 500-800 cells is enough to tell the differences. 2) Previously, we evaluated high resolution images of over ten cells in Fig.3B. In the revised version, we added the data of another twenty cells to Fig.3B. • How many times the experiments were repeated?We have repeated each experiment at least three times. After getting repeatable data, we stopped repeating. • At figure 6A, the authors stated that n>5 without stating what is the sample size exactly and if there is discrepancy between groups. While this is a valid way to represent sample size, however if you are going to statistically compare between them using One Way ANOVA, the exact sample size in each group should be reported. This again was observed in Fig. 4,5 and 7. Response to reviewer #2-12-1: Thanks for raising this question. We have repeated these experiments more than ten times. We have checked our original data, and we found we showed 4-5 times data here. Each group included 7-8 images.To solve the previous issues, meticulous revision needs to be done to the figures and the legends. I also recommend using dot plot figure whenever possible instead of bar graph to show the individual values and adding the mean and the SEM values in the text specially with the polarization ratios.Response to reviewer #2-12-2: Thanks for pointing these questions. We have used dot plot figures to improve the figure in the revised manuscript. In some parts, we have used bar plots. As we have several repeats in each group and protein levels and RNA levels data match with each other, we have used bar plots in some parts.3- A previous study in 2012 has found that "IDO Expression in Brain Tumors Increases the Recruitment of Regulatory T Cells and Negatively Impacts Survival". Based on this study findings, how the authors can explain this? Response to reviewer #2-13: Thanks for mentioning the paper from Dr. Maciej group. We have cited this in the revised version. IDO-1 is widely expressed in human tumors, including pancreatic cancers, HCC et al., and immune infiltrates in tumors [please refer to our reviewer and editor only Figure-2-3]. It is well known that IDO-1 expression in tumors recruits regulatory T cells. We also observed that some malignant cells in GBMs are highly expressed IDO-1 consistent with Dr. Maciej group [Fig.1E]. In this work, IDO-1 expression in neoplastic cells is our positive control. The topic of IDO-1 in brain neoplastic cells is not the scope of this study. Unfortunately, most IDO-1 inhibitors in cancer trials failed . In my personal view, we need to find an answer to whether IDO-1 expression neoplastic cells or immune cells in solid tumors significantly contribute to immune suppression or the creation of immune desert in solid tumors by recruiting Tregs or MDSA, including pancreatic cancers, glioma et al. We have been searching for an answer to this question for several years. You might have a chance to see our work soon . Minor Considerations:1- Please review the references (1-4) as they do not match the text in the first paragraph. While the text mentions on IDOs, these references describe the microglia role in Parkinson's disease and the polarization of macrophage.Response to reviewer #2-15: Thanks for pointing this out. It seems that it is a problem of the software which we used. We have freshened the citation in the revised version. 2- In the legend of figure 1, the authors need to describe which is murine brain and which is human brain as the figures should be able to stand by itself.Response to reviewer #2-16: Thanks. The murine brain is the mouse brain. We have specified in the revised version.3- Figure 1E is not explained either in the results section nor the figure legendResponse to reviewer #2-17: This is an inadvertent error. This error is also noticed by reviewer #2. We mixed the labeling in the previous version and corrected it in the revised version. 4- Fig. 2, the statistical significance was not demonstrated in some graphs.Response to reviewer #2-18: We have added the statistics to all images in the revised version.5- The manuscript needs to be revised for typo error like • Page 17 line 14, However However (delete one of them)Response to reviewer #2-19: Thanks. We have corrected it in the revised version. • Page14 line 20, Adipogenic? (Should be Adipogen?)Response to reviewer #2-20: Thanks. It is an inadvertent spelling mistake. We corrected it in the revised version.References:Antonios, J. K., Z. Yao, C. Li, A. J. Rao and S. B. Goodman (2013). "Macrophage polarization in response to wear particles in vitro." Cellular & Molecular Immunology 10(6): 471-482.Rostam, H. M., P. M. Reynolds, M. R. Alexander, N. Gadegaard and A. M. Ghaemmaghami (2017). "Image based Machine Learning for identification of macrophage subsets." Scientific Reports 7(1): 3521.Ruffinatti, F. A., T. Genova, F. Mussano and L. Munaron (2020). "MORPHEUS: An automated tool for unbiased and reproducible cell morphometry." Journal of Cellular Physiology 235(12): 10110-10115.Sevenich, L. (2018). "Brain-Resident Microglia and Blood-Borne Macrophages Orchestrate Central Nervous System Inflammation in Neurodegenerative Disorders and Brain Cancer." Frontiers in Immunology 9(697).Wainwright, D. A., I. V. Balyasnikova, A. L. Chang, A. U. Ahmed, K.-S. Moon, B. Auffinger, A. L. Tobias, Y. Han and M. S. Lesniak (2012). "IDO Expression in Brain Tumors Increases the Recruitment of Regulatory T Cells and Negatively Impacts Survival." Clinical Cancer Research 18(22): 6110.Reviewer #3: In this manuscript Ji et al investigated differential expression of IDO1 in microglia cells in the brain parenchyma, meninges, brain tumors and brain injury models. In addition, they also investigated potential function of IDO1 in microglia/macrophages. Below are some of the concerns that need to be addressed.1. Throughout the manuscript, please improve the writing. Logically explain why an experiment was done and what is the conclusion.Reviewer #3-1. Thanks. We have done our best to improve in the revised version. Please refer to the revised version. Previously, the manuscript is edited by Elsevier. 2. Rephrase the concluding paragraph in the introduction- it is confusing and hard to understand.Reviewer #3-2. Thanks. We rewrote the conclusion in the revised version. 3. Please explain why INF-Gamma was used- it is kind of choppy and it appears out of nowhere.Reviewer #3-3. Thanks for pointing this out. IFN-γ is a typical cytokine that increases IDO-1 expression and activity. Microglia/macrophage are more sensitive to IFN-γ treatment. Thus, we have used it to regulate IDO-1 expression. 4. Please include genetic inhibition of IDO1 and carry out some of the phenotypic data to provide additional evidence for IDO1 functions in microglia.Reviewer #3-4. Thanks for pointing out this. IDO-1 is an enzyme that is a druggable target. Both 1-MT and INCB24360 are well-tested candidates to inhibit IDO-1 activity in vivo . IDO-1 knockout mouse is commercially available now. We have checked the transcriptome data of microglia in IDO-1KO mice and found that deletion of IDO-1 does not significantly affect the expression of the typical M1 and M2 related markers . As TDO-2 is highly expressed in the brain, it is possible that TDO-2 is possible to substitute the function of IDO-1 after deleting IDO-1. In addition, INCB and I-MT, a well-tested IDO-1 inhibitor, can inhibit both IDO-1 and TDO-2. Thus, we have used the two potential drugs to inhibit IDO-1 activity. We know that genetic manipulation is an excellent way to provide more insight into IDO-1 function in microglia. However, the strength of our data is enough to support the conclusion in this paper. Reference(2018). Companies Scaling Back IDO1 Inhibitor Trials. Cancer Discov 8, OF5.Blair, A. B., Kleponis, J., Thomas, D. L., 2nd, Muth, S. T., Murphy, A. G., Kim, V., and Zheng, L. (2019). IDO1 inhibition potentiates vaccine-induced immunity against pancreatic adenocarcinoma. J Clin Invest 129, 1742-1755.Gonzalez-Pena, D., Nixon, S. E., Southey, B. R., Lawson, M. A., McCusker, R. H., Hernandez, A. G., Dantzer, R., Kelley, K. W., and Rodriguez-Zas, S. L. (2016). Differential Transcriptome Networks between IDO1-Knockout and Wild-Type Mice in Brain Microglia and Macrophages. PLoS One 11, e0157727.Han, X., Ma, L., Gu, J., Wang, D., Li, J., Lou, W., Saiyin, H., and Fu, D. (2021). Basal microvilli define the metabolic capacity and lethal phenotype of pancreatic cancer. J Pathol 253, 304-314.Hexige, S., Ardito-Abraham, C. M., Wu, Y., Wei, Y., Fang, Y., Han, X., Li, J., Zhou, P., Yi, Q., Maitra, A., et al. (2015). Identification of novel vascular projections with cellular trafficking abilities on the microvasculature of pancreatic ductal adenocarcinoma. J Pathol 236, 142-154.Le Naour, J., Galluzzi, L., Zitvogel, L., Kroemer, G., and Vacchelli, E. (2020). Trial watch: IDO inhibitors in cancer therapy. Oncoimmunology 9, 1777625.Long, G. V., Dummer, R., Hamid, O., Gajewski, T. F., Caglevic, C., Dalle, S., Arance, A., Carlino, M. S., Grob, J. J., Kim, T. M., et al. (2019). Epacadostat plus pembrolizumab versus placebo plus pembrolizumab in patients with unresectable or metastatic melanoma (ECHO-301/KEYNOTE-252): a phase 3, randomised, double-blind study. Lancet Oncol 20, 1083-1097.Ma, L., Hu, B., Liu, Y., Vermilyea, S. C., Liu, H., Gao, L., Sun, Y., Zhang, X., and Zhang, S. C. (2012). Human embryonic stem cell-derived GABA neurons correct locomotion deficits in quinolinic acid-lesioned mice. Cell Stem Cell 10, 455-464.Martinez, F. O., and Gordon, S. (2014). The M1 and M2 paradigm of macrophage activation: time for reassessment. F1000Prime Rep 6, 13.McWhorter, F. Y., Wang, T., Nguyen, P., Chung, T., and Liu, W. F. (2013). Modulation of macrophage phenotype by cell shape. Proc Natl Acad Sci U S A 110, 17253-17258.Pilli, D., Zou, A., Tea, F., Dale, R. C., and Brilot, F. (2017). Expanding Role of T Cells in Human Autoimmune Diseases of the Central Nervous System. Front Immunol 8, 652.Editor and Reviewer only Figure-1 Fig.1 IDO1 and TDO were highly expressed in human PDAC tissue.A, IDO1 expression pattern in precancerous pancreatic tissues, primary tumor and invasive lesions. B. IDO-1 expression in extrusion cells.Editor and Reviewer only Figure-2. IDO-1 is highly expressed in M1-like macrophage in the invasive region or metastatic site of pancreatic cancersA. The representative images of E-cadherin, IDO-1, and CD45RA staining in pancreatic cancer tissues and the lymph nodules . N, 10. B. Comparing counts of immune cells with IDO-1 higher in lymph nodules with metastatic neoplastic cells with that in lymph nodules without metastatic neoplastic cells C. Calibrating the size of IDO-1 high and CD45RA weak or IDO-1 low and CD45RA high cells (the longest diameter of cells). Student t-test.E. The representative images of CD11B and CD45RA staining in pancreatic cancer tissues and the lymph nodule . Patient number, 6. F. Calibrating the size of CD11B high and CD45RA weak or CD11B high and CD45RA high cells (the longest diameter of cells). Student t-test.G. Representative image of CD16 and IDO-1 staining in pancreatic cancer tissues . N, 6.Reviewers and Editor Only Figure -3A. Dextran uptake in RAW264.7 detected by Flow cytometry.Reviewer and editor only figure 4. A. The immunofluorescent staining control in mouse .B. IDO-1 and IBA-1 antibody immunostaining in human brain cortex .AttachmentResponse to reviewer and editors.docSubmitted filename: Click here for additional data file. 9 Aug 2021PONE-D-21-06075R1Characterization of IDO-1 expressing macrophages/microglia in the meninges and perivascular space of the human and murine brainPLOS ONEDear Dr. Saiyin,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.Considering the nature of the data presented in your manuscript, in our assessment, the title of this manuscript is too broad and unsupported by the evidence, and hence can be misleading. Only Figure. 1 has data supporting of expression of IDO-1 in the meninges and perivascular space of the human and murine brain. Rest of the data in the paper describe the effect of inhibiting IDO-1 in RAW264.7/BV-2 cells. While these cells can offer good system to study effect of IDO-1 inhibition, the findings from these cells cannot be meaningfully extrapolated to microglia in vivo.<o:p></o:p>Therefore, we ask that you modify title such that the presented evidence in the manuscript adequately supports it. In modifying your title, we specifically ask that the RAW264.7/BV-2 cells be identified in the title itself. The current title warrants additional in vivo studies examining effect of IDO-1 inhibition and its physiological implications.  We look forward to receiving your revised manuscript.Kind regards,Nagaraj KerurAcademic EditorPLOS ONE[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: All comments have been addressedReviewer #2: All comments have been addressedReviewer #3: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The authors have made all changes necessitated by the first round of review process. All additional data presented by the authors fully support the authors conclusion. I have no further comments or concerns.Reviewer #2: The authors addressedl the main concerns. Although this paper could be supplemented with stronger evidences, the theory presented opens new scopes in this field for further explorations.Reviewer #3: The authors have done a good job of addressing the questions raised by the reviewers. However, I felt the authors were adamant and stubborn in their responses. I wish they could use their wisdom to respond the questions raised by the reviewers.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  10 Aug 2021Response to editor and reviewersThank you for reviewing and revising our manuscript. Based on your suggestions comments, we have made the revision again. We all hope that our re-revised version fully addresses your and reviewers’ concerns and meets PLOS ONE’s publication criteria. If you have any further concern, we are willing to address as soon as possible. Editor comments and response to the concerns:Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.Considering the nature of the data presented in your manuscript, in our assessment, the title of this manuscript is too broad and unsupported by the evidence, and hence can be misleading. Only Figure. 1 has data supporting of expression of IDO-1 in the meninges and perivascular space of the human and murine brain. Rest of the data in the paper describe the effect of inhibiting IDO-1 in RAW264.7/BV-2 cells. While these cells can offer good system to study effect of IDO-1 inhibition, the findings from these cells cannot be meaningfully extrapolated to microglia in vivo.Therefore, we ask that you modify title such that the presented evidence in the manuscript adequately supports it. In modifying your title, we specifically ask that the RAW264.7/BV-2 cells be identified in the title itself. The current title warrants additional in vivo studies examining effect of IDO-1 inhibition and its physiological implications. We look forward to receiving your revised manuscript.Response to Dr. Kerur:Thank you for taking the time to review our manuscript rigorously. We all greatly appreciate your constructive suggestions and comments. We have re-revised our manuscript based on your and reviewer comments. We all wish our revision fully meets PLOS ONE’s publication criteria.We totally agree with your suggestion and comment that including BV-2 and RAW264.7 in the manuscript title. As you mentioned, our data in this manuscript did not fully support our previous title. The new title is “Characterizing the distributions of IDO-1 expressing macrophages/microglia in human and murine brain and evaluating the immunological and physiological roles of IDO-1 in RAW264.7/BV-2 cells”. Our title is flexible. If you have any good suggestion about our title, we are willing to accept or modify based on your suggestion.Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: All comments have been addressedThanks again.Reviewer #2: All comments have been addressedThanks again.Reviewer #3: All comments have been addressedThanks again.2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.Reviewer #1: YesReviewer #2: YesReviewer #3: Yes3. Has the statistical analysis been performed appropriately and rigorously?Reviewer #1: YesReviewer #2: YesReviewer #3: Yes4. Have the authors made all data underlying the findings in their manuscript fully available?The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.Reviewer #1: YesReviewer #2: YesReviewer #3: Yes5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: Yes6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The authors have made all changes necessitated by the first round of review process. All additional data presented by the authors fully support the authors conclusion. I have no further comments or concerns.Dear reviewer #1: Thanks again. Your comments are helpful to better our manuscript. Reviewer #2: The authors addressed the main concerns. Although this paper could be supplemented with stronger evidences, the theory presented opens new scopes in this field for further explorations.Dear reviewer #2: Thanks for the insightful comments about our manuscript. Your concerns about this paper are also our primary concern in the future. After this pandemic is over, we will address the immunological role of IDO+ macrophage/microglia in meninges and perivascular space, even the immune homeostasis in the parenchyma in our subsequent work. We are preparing more in vivo experiments to address your and our concerns about IDO-1+ macrophages/microglia in the brain. Reviewer #3: The authors have done a good job of addressing the questions raised by the reviewers. However, I felt the authors were adamant and stubborn in their responses. I wish they could use their wisdom to respond the questions raised by the reviewers.Dear Dr. Mallikarjun Patil:Thanks for taking the time to review our manuscript. In this specific season, we try our best to revise our manuscript. The availability of core facilities, especially FACS, is limited because of some restrictions and regulations. As non-native speakers of English, it is a little difficult for us to fluently and smartly communicate with reviewers or editors as a native speakers in the scientific community. In the future, we will try our best to improve our communication skills in the scientific literature, and you will have a chance to a better work about meningeal immunity soon. If some responses are inappropriate, it might be caused by our communication skills but not our true intention. Thanks for raising these questions. We will try our best to respond to the comments of reviewer and editor with wisdom in the future.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 22 Sep 2021Characterizing the distributions of IDO-1 expressing macrophages/microglia in human and murine brains and evaluating the immunological and physiological roles of IDO-1 in RAW264.7/BV-2 cellsPONE-D-21-06075R2Dear Dr. SaiyinWe’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Nagaraj KerurAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 27 Oct 2021PONE-D-21-06075R2 Characterizing the distributions of IDO-1 expressing macrophages/microglia in human and murine brains and evaluating the immunological and physiological roles of IDO-1 in RAW264.7/BV-2 cells Dear Dr. Saiyin:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Nagaraj Kerur Academic EditorPLOS ONE


We aimed to determine factors associated with this non-adherence in this specific population.we conducted a cross-sectional study at the Mother and Child Center in Yaounde from August to October 2018. Delayed clinic appointment was referred to as defaulters. Non-adherence was measured during the 3 days preceding inclusion by self-reported method following quantitative (missing dosage of ART), qualitative (ART taken with a delay of more than 2 hours) and combined measure. A threshold of non-adherence > 20% was considered high, with p<0.05 statistically significant.overall, 195 out of 251 (77.7%) eligible adolescents were included, of which 56.9% were girls (sex-ratio = 4/3). The mean age was 16.8 ± 1.5 years. The rate of defaulters was 21.0%. Following quantitative approach, 33.8% were non-adherent. Using combined approach, we had 41.0%. This non-adherence was associated with duration on ART > 5 years , defaulters  and HIV Viral Load (VL) ≥ 40 copies/ml .at this reference pediatric center, 4 out of 10 adolescents aged 15-19 years on ART are non-adherent, driven by missing dosage of drug intake. Strategies for enhanced adherence for late age adolescents are therefore warranted, by prioritizing interventions on defaulters and duration on ART > 5 years. Non-adherence to ART is referred to as missing dosage of ART (quantitative measure) or ART taken with a delay of more than 2 hours  or both  during the 3 days preceding inclusion by self-reported method. Acquired Immunodeficiency Syndrome (AIDS) is the second cause of mortality in adolescents worldwide and the first in Africa , 2. HIV-A study conducted in Douala among HIV-infected adults at Laquintinie Hospital revealed that 49% were non-adherence . In thisThe aim of this study was to determine factors associated with non-adherence to ART among HIV-infected adolescents aged 15 to 19 years and followed-up at the MCC reference pediatric center in Yaounde. Specifically, this study aimed at measuring the association between sociodemographic, clinical, biologic, enhance adherence factors and non-adherence to ART.Study design: we carried out a cross-sectional study to determine factors associated with non-adherence to ART among adolescents receiving ART at the MCC from August to October 2018. The following points justify the choice of this study site: (a) the MCC is the first center of excellence in Cameroon specialized in monitoring Adolescents Living with HIV (ALWHIV) on ART; (b) it is the health facility having the highest number of ALWHIV on ART in Cameroon. Study population was adolescents aged 15-19 years who came for their routine follow-up.Sampling: we used a consecutive sampling. Adolescents were included based on the following criteria: (a) aged 15-19 years; (b) on ART for at least 6 months; (c) registered for ART monitoring in the study site; (d) HIV-disclosure status completed and (e) who have provided their consent/assent. The minimum sample size was estimated using the formula z2p (1-p)/e2; Where n= sample size, z= 95% confidence interval= 1.96, p= proportion of non-adherent= 64% [ent= 64%  and e= sData collection: after pretesting the questionnaire, data collection was done by the principal investigator during routine clinic attendance of adolescents in the study site. Each adolescent was seen only once. The average time for completing a questionnaire was 20 minutes. About 3 questionnaires were completing every day from Monday to Saturday. Non-adherence was measured during the 3 days preceding inclusion by self-reported method following 3 approaches: (a) quantitative (missing dosage of ART); (b) qualitative (ART taken with a delay of more than 2 hours) and (c) mixed . These approaches were already used in other studies [ studies -16. A thData analysis: statistic tests were performed to compare the characteristics of adherents and non-adherents. Quantitative variables were converted into qualitative variables that were analyzed using the chi-square test. Logistic regression was used to estimate Odds-Ratios and their confidence interval in univariate and multivariate analyzes. Associated variables in univariate analyzes with a significance level < 0.20 were considered eligible in the multivariate model, with the exception of factors that could be clearly considered as a consequence Variables whose response modalities discriminated less than 5% of the sample were not considered eligible for the initial model. Variables independently associated with a value of p <0.05 were retained in the final model. SPSS version 21 software was used for all statistical analyzes. All p-values < 0.05 were considered statistically significant.Ethical approval and consent to participate: the protocol of this study was approved by the Institutional Ethics Committee of the Catholic University of Central Africa (N° 2018/0775/CEIRSH/ESS/MSP). After obtaining consent of parents/guardians and assent of adolescents, a standardized questionnaire was used to collect sociodemographic, clinical, laboratory and enhance adherence strategies data.Data confidentiality: the confidentiality of these data was managed by a system of anonymity based on a code . The completed questionnaires were kept in a locked drawer in the office of the principal investigator. Data treated were stored in a digital file of which access was only possible through the use of the investigator´s password.Participants: of the of 251 eligible participants, 195 were included (77.7%) ( (77.7%) . Among tDescriptive data: the proportion of non-adherence to ART was 41.0% (80/195) for combined measure, with 33.8% (66/195) quantitative and 7.2% (14/195) qualitative  and were 2.09 times more likely to be non-adherent versus those with > 150 000 F CFA. In contrast, no association between adherent and non-adherent was reported regarding age group, sex, residence, level of education, schooling, sexuality and alcohol use  and were 2.47 times more likely to be non-adherent versus those on ART ≤ 5 years. Moreover, participants with detectable VL (VL ≥ 40 copies/ml) were significantly lower among non-adherent  and had 1.85 (1/0.54) times less likely to be non-adherent versus those with undetectable VL (VL < 40 copies/ml) .Participants whose HIV disclosure was done by health personnel were significantly higher among non-adherent  and where 2.06 times more likely to be non-adherent versus those disclosed by their parent/guardian. Regarding HIV knowledge, participants who scored ≥ 12/15 (10.2%) were significantly higher among adherent  and had 7.14 (1/0.14) times less chance of being non-adherent versus those with < 12/15 .Factors associated with non-adherence to ART in multivariate analysis: non-adherence to ART was associated negatively with defaulters  and duration on ART > 5 years , but positively with HIV VL ≥ 40 copies/ml  (21-0.83) .Key results: the proportion of non-adherence to ART among adolescents aged 15 to 19 years was 41.0% . This non-adherence was associated with monthly income household > 150 000 F CFA, HIV disclosure by health personnel, duration on ART > 5 years, HIV knowledge ≥ 12/15, defaulters and HIV VL ≥ 40 copies/ml.Limitations: the main limitation of this study was the measure of non-adherence through a self-reported questionnaire. This method is not the most sensitive for identifying non-adherent, due to possible memory bias [ory bias , 11, 13.ory bias . Anotherory bias .Interpretation: the proportion of non-adherence in this study was lower than performance observed in other studies where adherence was measured through pill count and VL [t and VL , 13, 17.t and VL .Factors identified in the literature such as duration on ART  were alsHIV disclosure by health personnel was not associated with non-adherence. This finding indicates that there are potential factors in multivariate analysis that influence negatively adherence to ART. The proportion of defaulters was high among non-adherent. This trend was not significant in bivariate analysis, but became significant in multivariate model. In contrast, protective factors such as good knowledge on HIV/AIDS and detectable VL remained unchanged in multivariate analysis. These results confirm the interest of multivariate analysis that takes into account interactions of potential confounding factors.External validity: this study used a simple random sampling and included about 3 out of 4 adolescents from the target population. Hence, the findings reported can be generalized among HIV-infected adolescents aged 15 to 19 years and who are followed-up at this referral pediatric center. Nevertheless, with the monocentric site of this study, it is not possible to draw conclusions on other referral pediatric centers in Cameroon.At the MCC in Yaounde, about 4 out of 10 adolescents aged 15-19 years on ART are non-adherent, driven by missing dosage of drug intake. Strategies for enhanced adherence for late age adolescents are therefore warranted, by prioritizing interventions on defaulters and duration on ART of greater than 5 years, while adolescents with detectable VL constitute a target with high adherence. Importantly, a combination method towards adherence assessment would be more productive in resource-limited settings. Such approach might contribute to long-term therapeutic success and smooth transition from pediatric to adult care in this specific population.Assessing non-adherence to antiretroviral therapy (ART) using quantitative methods is the gold standard;Adolescents living with HIV remain a high-risk group for non-adherence to ART.The first original finding in this study was the positive association between detectable HIV viral load (VL ≥ 40 copies/ml) and non-adherence to ART;The second original finding revealed was the duration on ART, particularly > 5 years, which appeared as a risk factor of non-adherence.


Natural products have played an invaluable role in drug development, as about 24 percent of approved drugs belong to natural products or their derivative . CompareYu et al. summarized the biological and chemical aspects of Aspergillus niger strains including their sources, BGCs, and secondary metabolites as well as biological properties and biosynthetic pathways. A. niger has become promising application products which possess a large number of cryptic biosynthetic gene clusters (BGCs) and produce various biomolecules as secondary metabolites with a broad spectrum of application fields including agriculture, food industry, and medicine. In the past 5 years, the number of new bioactive compounds from A. niger has been decreasing so that more efforts should be made to explore more sources for isolation of new A. niger strains and to awaken their silent BGCs to manufacture novel functional biomolecules using new strategies.A review article by Chen et al. discovered the fusarisetins E and F produced by a Mangrove endophytic fungus Fusarium sp. 2ST2 from the healthy leaves of Kandelia candel which showed significant cytotoxicity against human A549 cell lines with IC50 values of 8.7 and 4.3 μM, respectively. At the same time, other new compounds including one new chromone fusarimone A, two new benzofurans fusarifurans A and B, three new isocoumarins fusarimarins A–C were isolated, which expanded the microbial secondary metabolite pool. Lu et al. also isolated novel secondary metabolite foeniculin K with cytotoxic activity from endophytic fungus Diaporthe foeniculina. Unfortunately, the other ten isolated analogues foeniculins I-J did not show cytotoxic activity. Their structures were established on the basis of 1H and 13C NMR spectra together with COSY, HSQC, HMBC, and NOESY experiments. Modern pharmacological research has revealed that Dendrobium huoshanense has anti-inflammatory, cytotoxic, hypoglycemic and antioxidant activity. Zhu et al. studied on an endophytic Streptomyces sp. HS-3-L-1 isolated from the leaves of Dendrobium huoshanense, and isolated three unique polyketide dimers with the cytotoxicity against MV4-11 human leukemia cell. So far only two similar natural products, strepolyketides B and C .As one of the diseases with high mortality, cancer is one of the focuses of new drug research and development.  B and C  were recDing et al. focused on the polycyclic tetramate macrolactams (PTM), and revealed that biosynthetic gene clusters (BGCs) are widespread in both Gram-positive and Gram-negative bacteria. In this study, they investigated a sponge endosymbiont Actinoalloteichus hymeniacidonis harboring a potential PTM-BGC. Xanthobaccin A as well as two previously reported tetramates, equisetin and ikarugamycin, exhibited antibacterial activities against Bacillus subtilis. In addition, these three tetramates were confirmed as metallophores for the first time. They found that all three tetramates could reduce ferric into ferrous iron, which triggers the Fenton chemistry reaction, and their antimicrobial mechanism is possibly mediated through Fenton chemistry.With the widespread use of antibiotics in clinic, bacteria gradually developed drug resistance, and this is a serious threat to the health and safety of the world. Therefore, it is of great significance to discover novel antibiotics. An original article by Wang et al. reported that three new humulane-type sesquiterpenoids, penirolide A, penirolide B, and 10-acetyl-phomanoxide, together with three known compounds aurasperone A, pughiinin A, and cyclo (L-Leu-L-Phe) from the endophytic fungus Penicillium sp. derived from the leaves of Carica papaya L. And four compounds penirolide B, 10-acetyl-phomanoxide, pughiinin A, and cyclo (L-Leu-L-Phe) can significantly inhibit glucagon-induced hepatic glucose production, with EC50 values of 33.3, 36.1, 18.8, and 32.1 μM, respectively. In response to glucagon, cAMP is a second messenger to initiate glucagon signaling cascades in hepatic glucose production. The treatment of these compounds suppressed cAMP accumulation indicated that they inhibited hepatic glucose production by suppression glucagon-induced cAMP accumulation. Liu et al. reported six new phthalan derivatives cytorhizophins D-I as well as three known derivatives cytorhizophin C, pestacin and rhizophol B from endophytic fungus Cytospora rhizophorae. Among them, cytorhizophins D-E and F-G were two pairs of diastereoisomers, all of them featuring a 1-phenyl-1,3-dihydroisobenzofuran scaffold with a highly oxygenated O-linked isopentenyl unit, and cytorhizophins H-I represent the first examples of phthalide family with fascinating 6/6/6/5 tetracyclic ring system fusing as unprecedented furo xanthen-2 (10bH)-one skeleton. Cytorhizophins D-E and F-G showed significant DPPH radical scavenging activities with EC50 values ranging from 5.86 to 26.80 μM, which are much better than that of the positive control ascorbic acid, they may be the promising lead compounds for the development of more effective antioxidants. Jia et al. focused on the mangrove-derived endophytes which are rich in bioactive secondary metabolites with a variety of biological activities. They isolated a fungus Pseudofusicoccum sp. J003 from mangrove species Sonneratia apetala Buch.-Ham. And then they identified a new sesquiterpenoid named acorenone C, two alkaloids, four phenolic compounds, and four steroid derivatives from this endophytic strain. Among them, acorenone C showed mild AChE inhibitory activity, with an inhibition rate of 23.34% at the concentration of 50 μM.Endophytic bacteria is a valuable resource pool of microorganisms with wide distribution, diverse species and diverse biological functions, and is an important source of novel compounds. In this topic, Wang et al. work reported that two new alkaloids tryptoquivaline Y and pseurotin I, together with eight known compounds, were isolated from Aspergillus felis FM324, and the fungus were purified from Hawaiian beach soil sample. One of the compounds showed weak antibacterial activity against Staphylococcus aureus, methicillin resistant Staphylococcus aureus and Bacillus subtilis. Two compounds inhibited NF-κB with IC50 values of 26.7 and 30.9 μM, respectively.Wu et al. research selected the appropriate mutant Aspergillus terreus ASM-1 through the chemical mutagenesis of A. terreus ML-44 and resulted in the isolation of three new prenylated aspulvinones V–X, together with analogs, aspulvinone H, J-CR, and R. All the compounds were evaluated for α-glucosidase inhibitory effects with acarbose as positive control. The results showed that aspulvinones V and aspulvinone H exhibited potent α-glucosidase inhibitory activities with IC50 values of 2.2 and 4.6 µM in mixed-type manners and aspulvinone H significantly suppressed the increases in postprandial blood glucose levels in the C57BL/6J mice. The results suggested that aspulvinones could be promising candidates for further pharmacologic research and the mechanism of the mutagenesis of the strain ASM-1 from strain ML-44 deserve further investigation which may make contribution to understanding the metabolic regulation of aspulvinones biosynthesis.Li et al. focused on the preparation of ilamycin F, a major secondary metabolite isolated from the marine-derived mutant strain Streptomyces atratus SCSIO ZH16 ΔilaR which were used as a scaffold to semi-synthesize eighteen new ilamycin derivatives (ilamycin NJL1–NJL18). Their study revealed that four of ilamycin NJLs have slightly stronger anti-TB activities against Mtb H37Rv  compared with that of ilamycin F on day 14th, but obviously display more potent activities than ilamycin F on day third, which means that these derivatives have fast-onset effect. In addition, most ilamycin NJLs had low cytotoxicity except ilamycin NJL1. These findings will promote the further exploration of structure-activity relationships for ilamycins and the development of anti-TB drugs.Tuberculosis (TB) is still a global disease threatening people’s lives. Ilamycins are novel cyclopeptides with potent anti-TB activities, Deng et al. discovered three sulfur-containing granaticin congeners, mycothiogranaticins A, B and granaticin MA from a granaticin-producing strain of Streptomyces vietnamensis GIMV4.0001. Gene disruptions suggested that the biosynthesis of mycothiogranaticins is mycothioldependent, providing experimental evidence for the biological origin of sulfur in this category of sulfurcontaining polyketides. And mycothiol was found to be involved in positive regulation of the biosynthesis of granaticins by maintaining the cellular redox balance. This is the first report that mycothiol can not only be a building block of polyketides but also play a regulatory role in the polyketide biosynthesis. Based on previous research that P450 Astb can dually oxidize two methyl groups (C-19 and C-21) of preasperterpenoid A to asperterpenoid A with 3-carboxyl and 11-hydroxymethyl groups, Huang et al. confirmed the oxidation order of C-19 and C-21 catalyzed by AstB, by using the combination of the quantum chemistry calculations and the experiments of obtaining the potential intermediates and the HPLC-MS detection of the potential intermediates. In the end, they revealed the catalyzed order of AstB in asperterpenoid A biosynthesis and the relationship between the oxidation stations of C-19 and C-21 in asperterpenoids and their mPTPB inhibition.This special issue also covers some biosynthesis research. In summary, the above works presented in this special research topic illustrate the diversity of microbial natural products and highlight the importance of developing new methods to impulse the discovery of new compounds. Microorganisms are still the treasure house of new drug development in the future.